It is proposed to investigate the biosynthesis of two naturally occurring nucleosides that exhibit potentially important biological activity and unusual structures. The first nucleoside whose biosynthesis will be investigated is sinefungin, which is produced by Streptomyces griseolus and S. incarnatus. Sinefungin possess antifungal, antiprotozoal, and antiviral activity, and has recently been found to be active against the AIDS virus. Previous investigations have shown that sinefungin is derived from L-ornithine via L-arginine and that C-C bond formation to C-5 of ornithine occurs with overall inversion of configuration. Pyridoxal phosphate appears to be required for this reaction. The identity of the other partner in the bond forming process has not yet been clearly defined, since both ATP and adenosine have been shown to be catabolized in cell-free extracts of S. griseolus that synthesize sinefungin. Future investigations will have several goals. These include at least partial purification of the C-C bond forming enzyme from the cell-free extracts, identification of the second partner in the bond forming process, and determination of the stereochemistry of C-C bond formation at the second reaction terminus. If highly purified enzyme can be obtained, other goals will be to fully characterize the protein and to examine the mechanistic role played by pyridoxal phosphate in the C-C bond-forming reaction. The possibility of using the purified enzyme to prepare sinefungin analogs would also be explored. The second nucleoside whose biosynthesis will be investigated is griseolic acid A and its congeners. These substances are potent inhibitors of cAMP phosphodiesterase and potential "cardiotonic" agents produced by S. griseoaurantiacus. It appears that the biosynthesis of these compounds may proceed by C-C bond formation to C-5' of an adenylyl moiety. The findings with respect to sinefungin biosynthesis may therefore provide some insight into the biosynthesis of the griseolic acids. The structures of the griseolic acids pose three significant biosynthetic questions. The first concerns the nature of the primary building blocks that are involved in the formation of the molecules. The second question concerns the mechanism of C-C bond formation to C-5' of a putative adenylyl building block. Finally, there is the question of the mechanism of formation of the ether linkage present in three of the griseolic acids. The ether linkage appears to be required for the observed biological activity of the griseolic acids, since the one congener which lacks this functionality, griseolic acid D, is not an inhibitor of cAMP phosphodiesterase. The answers to these questions will initially be sought by administration of appropriate labeled precursors to whole cell cultures. These studies will then be followed up with efforts to prepare and characterize a cell-free extract of S. griseoaurantiacus that will synthesize the griseolic acids.